Apoptosome Structure and Procaspase Activation Abstract Programmed cell death pathways have evolved in eukaryotes to provide appropriate responses to developmental cues, environmental stress, cellular pathogens and genomic insults. These death pathways also provide a line of defense against unrestricted cell proliferation. Hence, apoptosis plays a central role in the maintenance of human health, while regulating tissue development and homeostasis. In this renewal, we will continue our combined structural and biochemical studies of apoptosomes. These platforms assemble from Apaf-1 like proteins and activate procaspases (pc) in the intrinsic cell death pathway. Apaf-1 CARDs and pc-9 CARDs form an activation disk that sits above the central hub of the wheel-like apoptosome. The disk and a conserved linker in pc-9, play important roles in the activation of pc- 9 catalytic domains, which bind to the central hub. Together, disk assembly and bound pc-9 catalytic domains create an asymmetric proteolysis machine. We have also shown that caspase-3 down-regulates pc-9 activity through a novel feedback mechanism which uses overlapping binding sites on the hub. In Aim 1, cryo-EM structures will be determined of the human apoptosome in complexes with pc-9 and caspase-3. These structures will provide a framework for biochemical studies on the mechanisms underlying assembly, activation and execution in programmed cell death. In addition, crystallization experiments will target interactions between regulatory beta-propellers and cytochrome c that kick-start assembly. In Aim 2, a structure of the Drosophila apoptosome will be determined at near atomic resolution. A 3D structure of the Dark apoptosome with Dronc, its initiator procaspase will also be determined. This data will be used to carry out targeted mutagenesis and biochemical experiments to understand the function of Dark. Intriguingly, we find that the geometry of active site CARDs differs in the ground states of human, fly and worm apoptosomes. In a unifying hypothesis, we propose that initiator procaspases in all metazoans will form a CARD-CARD disk and be activated by proximity induced association of catalytic domains with their apoptosome partners. When taken together, these Aims should reveal new aspects of apoptosome function that are germane to human health and disease.